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Journal of Zhejiang University (Agriculture and Life Sciences)  2018, Vol. 44 Issue (6): 743-747    DOI: 10.3785/j.issn.1008-9209.2017.08.152
    
Prokaryotic expression of extracellular domain of avian CD133 protein and preparation of polyclonal antibody against CD133
ZHANG Qiaoyan1, SHAO Fengjin1, YU Xiangqian2, TAN Xun1*
(1. College of Animal Sciences, Zhejiang University, Hangzhou 310058, China; 2. Pudong Center of Animal Disease Prevention and Control, Shanghai 201200, China)
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Abstract  To construct the prokaryotic expression vector of avian CD133 extracellular region, and develop an antiavian CD133 polyclonal antibody, a peptide fragment with 89 amino acids at the N terminal of CD133 protein was selected as a candidate antigen based on bioinformatic analysis. The DNAsequence of the selected peptide fragment was synthesized from lung tissue cDNA by polymerase chain reaction (PCR) and subcloned into an expression plasmid pET-28a (+ ) and propagated in Escherichia coli strain BL21 (DE3). Protein expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG). The target protein was purified by using Ni gravity columns and used to immunize BALB/c mice. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed high expression of recombinant CD133 protein in E. coli strain BL21. Western-blotting assay demonstrated that the blood serum derived from the immunized mice specifically probed recombinant CD133 on cellulose acetate membrane. Immunohistochemical assay showed that the anti-avian CD133 serum developed in this study could detect CD133-positive cells in the endothelium of avian pulmonary arteries. In conclusion, the present study validates the antigenicity of the N terminal extracellular domain of avian CD133 molecule, and suggests that the recombinant CD133 protein can be used to develop anti-avian CD133 polyclonal antibody. 


Key wordsCD133 protein      polyclonal antibody      avian     
Published: 25 November 2018
CLC:  Q 786  
  S 858.31  
Cite this article:

ZHANG Qiaoyan, SHAO Fengjin, YU Xiangqian, TAN Xun. Prokaryotic expression of extracellular domain of avian CD133 protein and preparation of polyclonal antibody against CD133. Journal of Zhejiang University (Agriculture and Life Sciences), 2018, 44(6): 743-747.

URL:

http://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2017.08.152     OR     http://www.zjujournals.com/agr/Y2018/V44/I6/743


禽CD133 胞外结构域原核表达及多克隆抗体的制备

为构建禽CD133 胞外区原核表达载体,制备抗CD133 多克隆抗体,在生物信息学分析的基础上,选取禽CD133 蛋白N端胞外区一段由89 个氨基酸残基构成的肽段作为候选抗原,通过聚合酶链式反应(polymerase chainreaction, PCR)扩增其编码基因,亚克隆至pET-28a(+)表达载体,转化大肠埃希菌BL21(DE3)感受态细胞,用异丙基β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside, IPTG)诱导表达;目的蛋白经镍柱纯化后免疫BALB/c小鼠,制备抗血清。结果表明,构建的原核表达载体可高效表达CD133 重组蛋白。蛋白质印迹法(Westernblotting)证实鼠抗禽CD133 血清可特异性识别CD133 重组蛋白。免疫组化染色结果显示,该抗血清可用于检测组织中的CD133 阳性细胞。上述结果表明,利用禽CD133 蛋白N端胞外结构域重组蛋白为抗原成功制备出了抗禽CD133 多克隆抗体。

关键词: CD133 蛋白,  多克隆抗体,  家禽 
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